Summary

All animal embryos pass through a stage during which developmental control
is handed from maternally provided gene products to those synthesized from the
zygotic genome. This maternal-to-zygotic transition (MZT) has been extensively
studied in model organisms, including echinoderms, nematodes, insects, fish,
amphibians and mammals. In all cases, the MZT can be subdivided into two
interrelated processes: first, a subset of maternal mRNAs and proteins is
eliminated; second, zygotic transcription is initiated. The timing and scale
of these two events differ across species, as do the cellular and
morphogenetic processes that sculpt their embryos. In this article, we discuss
conserved and distinct features within the two component processes of the
MZT.

Introduction

“What's past is prologue”Shakespeare, The Tempest (II.i)

The maternal genome controls virtually all aspects of early animal
development. Maternal mRNAs and proteins, which are loaded into the egg during
oogenesis, implement basic biosynthetic processes in the early embryo, direct
the first mitotic divisions, and specify initial cell fate and patterning. As
development proceeds, two processes are triggered that together form the
maternal-to-zygotic transition (MZT): first, a subset of the maternal mRNAs is
eliminated; second, the transcription of the zygotic genome begins. Initially,
the destruction of maternal mRNAs is accomplished by maternally encoded
products. However, zygotic transcription leads to the production of proteins
and microRNAs (miRNAs) that provide feedback to enhance the efficiency of
maternal mRNA degradation. In addition, among the earliest mRNAs synthesized
de novo in the embryo are transcriptional activators that enhance the
efficiency of zygotic transcription. The net result is that the control of
development is transferred from the maternal to the zygotic genome.

The purpose of this primer is to review the events that constitute the MZT
in representatives of different branches of the metazoan (see Glossary,
Box 1) evolutionary tree in
which the MZT has been most extensively studied
(Fig. 1): echinoderms
(Strongylocentrotus purpuratus), nematodes (Caenorhabditis
elegans), insects (Drosophila melanogaster), fish (Danio
rerio), amphibians (Xenopus laevis) and mammals (Mus
musculus). The MZT also occurs in plants, but this is not considered here
(reviewed by Baroux et al.,
2008). We begin with an overview of the major developmental events
that occur during the MZT. We then examine what is known about maternal
transcript destabilization and the subsequent activation of the zygotic
genome. Our focus is on the dynamics and the scale of these events, their
underlying molecular mechanisms and their functions.

A comparative overview of the maternal-to-zygotic transition (MZT) in
several model organisms. Key embryonic stages for each model organism are
depicted schematically above the corresponding cleavage cycle and time after
fertilization. The red curves represent the degradation profiles of
destabilized maternal transcripts in each species. The light and dark blue
curves illustrate the minor and major waves, respectively, of zygotic genome
activation. The last embryonic stage presented for each organism is the
developmental point at which there is a major requirement for zygotic
transcripts.

Box 1. Glossary

Cis-element

A defined DNA or RNA region that mediates transcription in the case of
DNA, or processes such as translation, stability, export and localization in
the case of RNA. Cis-elements are bound by transfactors, which can be proteins
or RNAs

Compound chromosomes

Chromosomes with their arms reassorted relative to their centromeres.
For example, for a metacentric chromosome that normally has a left arm (L) and
a right arm (R), giving the composition L:R, compound chromosomes might have
two right arms attached to one centromere (R:R) and two left arms attached to
another centromere (L:L). A diploid cell would still have two left arms and
two right arms. However, meiosis can result in gametes (and thus, after
fertilization, embryos) that lack both left arms or both right arms. More
complex compound chromosomes also exist.

Gap phase

A pause in the cell cycle between DNA synthesis (S phase) and mitosis
(M phase). The gap between M and S phase is termed G1, and that between S and
M phase, G2.

Gene ontology (GO) analysis

Classification system whereby genes that encode products contributing
to a common cellular component, or implementing a particular molecular or
biological function, share a corresponding `GO term' designating them as such.
This facilitates the bioinformatic identification of trends, in which genes of
a particular class are similarly regulated.

Male pronuclear remodeling

The morphological and biochemical modifications, such as the
replacement of protamines with histones, that occur after fertilization and
that transform a dormant sperm nucleus into a functional male
pronucleus.

Metazoan

A eukaryotic multicellular animal (excluding single-celled protozoa and
multicellular sponges). Metazoa are subdivided into Bilateria (animals with
bilateral symmetry) and Radiata (animals with radial symmetry). Bilateria can
be further subdivided, based on particular developmental distinctions, into
deuterostomes (e.g. echinoderms, such as sea urchins, and chordates, such as
tunicates and vertebrates) and protostomes (e.g. Ecdysozoa, such as arthropods
and nematodes).

Parthenogenesis

The development of an egg without fertilization, common in some insects
and arthropods.

Protamines

Small, basic, arginine-rich proteins that are often found associated
with DNA in sperm nuclei.

Syncytial divisions

Nuclear divisions without cytokinesis. Syncytial divisions can occur
after fertilization in the common cytoplasm of some early embryos, notably in
many insects.

Trans-factor

A protein or small RNA that binds a cis-element and mediates some
aspect of nucleic acid regulation.

Stage directions: an overview of early embryogenesis

“We will draw the curtain and show you the
picture”Shakespeare, Twelfth Night (I.v)

To place the events of the MZT in context, we describe the setting in which
it occurs - activated eggs and early embryos - with a focus on major
developmental and cell cycle hallmarks.

Egg activation

Egg cells are suspended both in their metabolic activity and in their cell
cycle, the latter at a particular stage of meiosis that varies from species to
species. Egg activation comprises a multitude of events, triggered in response
to external stimuli, which bring the mature egg cell out of its suspended
state. Egg activation is necessary - and, in some cases, also sufficient - for
the initiation of embryogenesis (reviewed by
Horner and Wolfner, 2008). In
echinoderms, nematode worms and many vertebrates, fertilization is the trigger
for egg activation. In other species, such as insects that undergo
parthenogenesis (see Glossary, Box
1), fertilization is not required; instead, changes in the ionic
environment, pH, or mechanical stimulation trigger activation. In
non-parthenogenetic insects, such as D. melanogaster, egg activation
also occurs independently of, and prior to, fertilization, and involves a
combination of osmotic and mechanical stimulation. Egg activation always
results in a rise in intracellular calcium, waves of which initiate a
signal-transduction cascade that brings about the resumption of meiosis, male
pronuclear remodeling (see Glossary, Box
1) and cytoskeletal (and, for externally developing embryos,
eggshell) rearrangements, as well as alterations in gene regulation at both
the post-transcriptional and post-translational level.

Early cleavage divisions and the mid-blastula transition

In most metazoans, following the completion of meiosis and the fusion of
the male and female pronuclei, synchronous and rapid cell cycles ensue
(Fig. 1), often without gap
phases (see Glossary, Box 1).
In mammalian and nematode embryos, however, the early mitotic divisions are
asynchronous. In most species, complete or nearly complete cytokinesis follows
every cleavage. In D. melanogaster and many other insects, however,
the synchronous early cleavage cycles occur without cytokinesis, producing
nuclei that migrate through the yolk to the periphery of the embryo, where
they undergo additional synchronous syncytial divisions (see Glossary,
Box 1). The early mitotic
cycles range in length from 8 minutes in flies, to 15 minutes in zebrafish, to
12 hours in mice.

The early mitoses eventually produce a blastula or `ball of cells' (in most
amphibians and echinoderms) or a peripheral layer of blastoderm nuclei (in
many insects) that occupies the same volume of cytoplasm as the original
unfertilized egg (Fig. 1). In
echinoderms, X. laevis and zebrafish, the synchronous divisions are
followed by asynchronous cleavages, with the introduction of gap phases during
a developmental event termed the `mid-blastula transition' (MBT; see
Box 2). In D.
melanogaster, the MBT also involves the introduction of a gap phase,
which permits the invagination of plasma membrane between the syncytial
blastoderm nuclei to form the cellular blastoderm. The MBT is followed by the
onset of gastrulation, during which the germ layers of the embryo - endoderm,
mesoderm and ectoderm - form through a combination of cell migration,
ingression and invagination.

Act 1: maternal transcript destabilization

“If it were done, when `tis done, then `twere well It were done
quickly.”Shakespeare, Macbeth (I.vii)

The first event of the MZT is the elimination of maternal transcripts.
Here, we discuss the scale, mechanisms and functions of this process.

Dynamics and scale of maternal transcript destabilization

Estimates of the fraction of the protein-coding genome represented as
maternal mRNAs range from 40% in the mouse
(Wang et al., 2004), to 65% in
D. melanogaster (Lecuyer et al.,
2007; Tadros et al.,
2007), to 75% in S. purpuratus
(Wei et al., 2006). The
elimination of a subset of these transcripts is the first event of the MZT
(Figs 1 and
2). Destabilized mRNAs range
from 30% of maternal mRNAs in C. elegans
(Baugh et al., 2003), to 33% in
mice (Hamatani et al., 2004),
to 35% in D. melanogaster (De
Renzis et al., 2007); corresponding microarray-based data are
unavailable for echinoderms and amphibians. Notably, these transcripts remain
in oocytes for days, weeks or even months prior to their elimination, which
occurs in a matter of hours; hence, this represents a cataclysmic change in
the stability of a significant fraction of the maternal transcript pool.

Box 2. MZT versus MBT

The literature on early metazoan embryogenesis is rife with inconsistencies
and confusion regarding the terms `maternal-to-zygotic transition' (MZT) and
`mid-blastula transition' (MBT). Here, we use the following definition of the
MZT: the period that begins with the elimination of maternal transcripts,
continues through the production of zygotic transcripts and ends with the
first major morphological requirement for zygotic transcripts in embryonic
development. One key difference between this definition and those used
previously [for example, that adhered to by Baroux et al.
(Baroux et al., 2008)] is the
idea that the MZT spans a period rather than being a point in time. Another
important distinction relates to the starting point of the MZT, which we
propose is egg activation rather than fertilization. In D.
melanogaster, the elimination of maternal transcripts begins immediately
upon egg activation, prior to and independently of any zygotic input
(Bashirullah et al., 1999;
Tadros et al., 2003). This
also appears to be true in the majority of model organisms in which transcript
elimination has been studied (see text). Conversely, the term `MBT' was first
used to describe a developmental event in amphibian embryogenesis in which,
after the first 11 or 12 synchronous cleavage divisions, the cell cycle
lengthens, gap phases appear and mitoses become desynchronized
(Gerhart, 1980). Since then,
researchers have applied the term to analogous stages in other animals, such
as fish (Kane and Kimmel,
1993) and flies (Blankenship
and Wieschaus, 2001). It is important to note that, although the
MBT roughly coincides with the major activation of the zygotic genome in these
organisms, the same does not appear to hold true in mammals.

Transcript destabilization is achieved through the combined action of at
least two types of degradation activity. The first, `maternal', activity is
exclusively maternally encoded and functions upon egg activation in the
absence of zygotic products. The second, `zygotic', activity requires zygotic
transcription. In D. melanogaster
(Fig. 2A), owing to the
uncoupling of egg activation and fertilization, the maternal activity can be
observed separately from the zygotic one
(Bashirullah et al., 1999;
Tadros et al., 2003).
Genome-scale analyses have shown that over 20% of maternal transcripts are
destabilized by this maternal activity
(Tadros et al., 2007). The
zygotic activity further destabilizes these transcripts and eliminates an
additional 15% of maternal mRNAs, which results in the overall elimination of
35% of maternal transcripts by the end of the MZT
(De Renzis et al., 2007).

In the mouse, a large fraction of the maternally supplied mRNAs is degraded
by the two-cell stage (Piko and Clegg,
1982), and fertilization triggers at least some of this
destabilization (Alizadeh et al.,
2005). Gene expression profiling experiments have provided
evidence for what are probably the maternal and zygotic degradation activities
(Hamatani et al., 2004)
(Fig. 2B): some maternal
transcripts are degraded soon after fertilization (e.g. cluster 9); others
show a later and very rapid decrease that coincides with the major onset of
transcription at the two-cell stage (e.g. cluster 7). Genome-scale transcript
analyses in zebrafish (Fig. 2C)
(Ferg et al., 2007;
Mathavan et al., 2005) and
C. elegans (Baugh et al.,
2003) also show destabilization profiles that are consistent with
maternal and zygotic degradation activities.

Distinct groups of mRNAs are enriched in the stable versus unstable
maternal subsets. For example, in D. melanogaster, Gene Ontology (GO)
term analysis (see Glossary, Box
1) has shown that the stable subset is highly enriched for
transcripts related to RNA transactions (binding, metabolism, translation),
consistent with a requirement for these processes both during the MZT and
beyond. By contrast, unstable transcripts are enriched for GO terms related to
the cell cycle (Tadros et al.,
2007), the possible functional significance of which is discussed
in the section on the function of maternal transcript destabilization. In the
mouse, too, unstable maternal transcripts are enriched for GO terms related to
the cell cycle; however, GO terms related to RNA metabolism, RNA binding and
protein synthesis are enriched in zygotically synthesized transcripts rather
than in maternal transcripts (Hamatani et
al., 2004). This difference might reflect the very rapid early
development of fly embryos (requiring pre-loaded RNA and protein synthetic
machinery); in the more slowly developing mammalian embryo, there is plenty of
time for the zygotic production of these components.

Degradation profiles of maternal transcripts during the MZT.
Presented are profiles of degrading maternal transcripts (red) based on
published data from three different model systems. (A) Four subsets of
maternal transcripts have been characterized in D. melanogaster:
Stable mRNAs (e.g. rpA1; RpLP2 - FlyBase); mRNAs targeted
solely by the maternal (e.g. nos) or the zygotic (e.g. bcd)
degradation pathways; and those targeted by both (e.g. Hsp83)
(Bashirullah et al., 1999;
Surdej and Jacobs-Lorena,
1998). (B) In the mouse, genomic profiling
(Hamatani et al., 2004) has
revealed clusters of maternal transcripts that appear to degrade with kinetics
that correspond to the subsets identified in D. melanogaster:
Clusters 3 and 9 degrade in the absence of significant zygotic transcription;
degradation of cluster 7 transcripts appears to accelerate coincident with the
onset of the major zygotic genome activation (ZGA) wave, whereas cluster 6b
transcripts most resemble the stable subset. (C) In zebrafish embryos,
transcripts that degrade have been assigned to three different groups
according to whether the majority of their degradation occurs (a) before, (b)
during or (c) following ZGA (Mathavan et
al., 2005). In each case, the dotted black line denotes the timing
of the first major wave of ZGA.

Mechanisms of maternal transcript destabilization

The exact link between calcium signaling during egg activation and maternal
transcript destabilization remains unclear, in part because mutations that
affect calcium signaling are pleiotropic, which makes their recovery in
genetic screens based on maternal transcript stability rare
(Tadros et al., 2003).
Nonetheless, work in D. melanogaster has identified a
post-translational and post-transcriptional cascade triggered upon egg
activation that functions in transcript destabilization. Following egg
activation, the Pan gu (PNG) Ser/Thr kinase complex promotes the translation
of an RNA-binding protein, Smaug (SMG)
(Tadros et al., 2007). SMG
acts as a specificity factor, binding maternal transcripts that contain
cis-elements (see Glossary, Box
1), known as SMG recognition elements
(Smibert et al., 1996). SMG
recruits the CCR4/POP2/NOT-deadenylase complex to these target transcripts,
thus prompting the removal of their poly(A) tail, the first and rate-limiting
step in mRNA degradation (Semotok et al.,
2005; Semotok et al.,
2008). SMG is essential for eliminating the majority of unstable
maternal transcripts (Tadros et al.,
2007), although it is not yet clear what fraction of these is
directly bound by SMG.

In D. melanogaster, computational analyses have identified two
additional cis-elements that are enriched in destabilized maternal transcripts
(De Renzis et al., 2007): one
resembles PUF-family binding sites and could, in principle, be bound by
Pumilio (PUM), a post-transcriptional regulator implicated in both
translational repression and destabilization of mRNAs; the other resembles
AU-rich cis-elements (AREs), which mediate either transcript stabilization
(through binding of HuR proteins) or destabilization (through binding of
AUF1). To date, the potential roles of PUM and ARE-binding proteins in
maternal transcript destabilization in D. melanogaster have not been
effectively tested. In X. laevis, however, the ARE-mediated pathway
(Voeltz and Steitz, 1998)
works with the Embryonic Deadenylation Element Binding Protein (EDEN-BP)
(Paillard et al., 1998) to
trigger the deadenylation of maternal transcripts upon fertilization via the
recognition of two distinct types of AU-rich cis-elements. EDEN-BP is in a
complex with 158 mRNAs that are enriched for the EDEN cis-element
(Graindorge et al., 2008).
Intriguingly, the addition of calcium to cell-free extracts induces an
unidentified kinase-phosphatase cascade that results in both EDEN-BP
dephosphorylation and a concomitant increase in its deadenylation activity
(Detivaud et al., 2003). This
might explain how EDEN-BP, which is present at equivalent levels in oocytes
and early embryos (Paillard et al.,
1998), is activated in response to fertilization.

Notably distinct from the maternal degradation pathways in other systems,
fertilization-induced deadenylation in X. laevis does not trigger
decay until after the onset of zygotic transcription
(Audic et al., 1997;
Duval et al., 1990;
Voeltz and Steitz, 1998),
which indicates a requirement for a zygotically produced factor that acts
after deadenylation has taken place. However, in the absence of genome-wide
profiling data on transcript stability, it remains unclear whether this is the
general pattern for destabilized maternal mRNAs in X. laevis.

Temporal coupling of deadenylation and destabilization does occur in the
zygotic degradation pathway in X. laevis. Here, Cyclin A1
and Cyclin B2 transcripts are targeted for elimination through
discrete regions in their 3′UTRs
(Audic et al., 2001;
Audic et al., 2002). Although
the trans-factors that mediate this process are unknown, preliminary evidence
indicates that it might be accomplished through the binding of a miRNA
(Guo et al., 2008). Indeed,
mounting evidence suggests that miRNAs are mediators of the zygotic
degradation pathway. For example, the zygotic expression of miR-430
in zebrafish embryos is responsible for the clearance of several hundred
maternal messages (Giraldez et al.,
2006). miR-430 requires additional, zygotically produced,
non-miRNA factors to degrade a subset of its targets
(Ferg et al., 2007). The
D. melanogaster miR-309 family of miRNAs, which are synthesized
zygotically in a SMG-dependent manner, participate in the destabilization of
several hundred maternal mRNAs in a manner analogous to that of zebrafish
miR-430 (Benoit et al.,
2009; Bushati et al.,
2008).

Minor and major waves of zygotic genome activation during the MZT.
The numbers of transcripts produced in the minor (light blue) and major (dark
blue) waves of zygotic genome activation are presented. The schematics are
based on data from (A) D. melanogaster
(De Renzis et al., 2007),
(B) mouse (Hamatani et al.,
2004) and (C) zebrafish
(Mathavan et al., 2005).

Beyond these studies, it has been shown in D. melanogaster that
genomic loci that map to different chromosome arms are required for the
destabilization of distinct subsets of maternal mRNAs
(De Renzis et al., 2007).
Thus, it is likely that there are several distinct zygotic degradation
activities.

Functions of maternal transcript destabilization

In the absence of mutations that specifically abrogate maternal transcript
destabilization, it has been difficult to demonstrate functions for this
process in development - assuming there are any. One possibility is that the
elimination of a significant fraction of maternal mRNAs might be necessary to
prevent abnormal mRNA dosage in the embryo, representing the maternal
counterpart to the zygotic lethality caused by chromosomal segmental
aneuploidy (Lindsley et al.,
1972). If so, then the elimination of a significant fraction of
all maternal mRNAs - rather than the elimination of a specific maternal mRNA
(or set of mRNAs) - might be required.

Alternatively, the elimination of specific maternal mRNAs could be
essential for early development. For example, the elimination of ubiquitously
distributed maternal mRNAs might permit the patterned transcription of their
zygotic counterparts to direct spatially and temporally localized control.
Consistent with this hypothesis, genome-scale analyses have determined that
the zygotic transcripts that replace their uniformly distributed maternal
counterparts tend to be expressed in restricted patterns much more frequently
than does the average gene (De Renzis et
al., 2007). This particular role would be permissive rather than
instructive, as maternal transcript elimination would have no function other
than to permit patterned zygotic transcripts to exert their influence. For
example, maternal string mRNA, which encodes a D.
melanogaster homolog of the cell cycle regulator Cdc25, is eliminated
throughout the embryo by the end of the MZT, and is then replaced by patterned
zygotic expression that correlates with mitotic domains in the gastrulating
embryo (Edgar and O'Farrell,
1990; Foe, 1989).
The degradation of ubiquitous maternal cell cycle mRNAs might be required to
permit these patterned mitoses.

A third possibility is that the elimination of maternal mRNAs is
instructive rather than permissive. For example, a gradual decrease in
maternal cell cycle mRNA levels in early D. melanogaster embryos
might direct the gradual increase in mitotic cycle length and the pause at
interphase 14 that allows cellularization. Consistent with this hypothesis,
increasing or decreasing the maternal dosage of the string and
twine genes - and thus of maternal string and twine
mRNA levels - results in an increase or decrease, respectively, in the number
of nuclear cycles that occur prior to blastoderm cellularization
(Edgar and Datar, 1996). Also
consistent with this hypothesis, embryos produced by smaug mutant
females, in which cell cycle mRNAs such as Cyclin A and B
fail to be eliminated, continue to undergo very rapid nuclear cycles without
slowing and pausing as in wild-type embryos
(Benoit et al., 2009).

These hypotheses are not mutually exclusive. For example, the elimination
of maternal cell cycle mRNAs could be both instructive (slowing the cell
cycle) and permissive (allowing patterned mitoses after the MZT).

Act 2: zygotic genome activation

“Our remedies oft in ourselves do lie”Shakespeare, All's Well That Ends Well (I.i)

The second event of the MZT is the onset of zygotic transcription, commonly
referred to as zygotic genome activation (ZGA). Here we discuss the scale,
mechanism and function of ZGA.

Dynamics and scale of zygotic genome activation

ZGA occurs in successive waves of increasing degree (see Figs
1,
3). In terms of the embryonic
mitoses, mouse and sea urchin embryos begin ZGA the earliest, with the first
wave of transcription commencing at the one-cell stage. Whereas BrUTP has been
shown to be incorporated into the male pronucleus during the one-cell stage in
the mouse (Aoki et al., 1997),
genome-wide analyses have revealed only a single transcript, the production of
which is sensitive to an RNA polymerase II inhibitor, α-amanitin, at
this stage (Hamatani et al.,
2004). D. melanogaster initiates its minor and major
transcriptional waves during cleavage cycles 8 and 14, respectively. In terms
of absolute time, however, the very rapid cleavage cycles of the early fly
embryo mean that ZGA begins several hours earlier than in the mouse. In D.
melanogaster, transcription has been detected prior to the first wave of
ZGA: genome wide, 30 genes are predicted to be transcribed during the first
few nuclear cleavages, within the first 30 minutes after fertilization
(Lecuyer et al., 2007).

In D. melanogaster, the strictly zygotic mRNA set is highly
enriched for transcription factors (De
Renzis et al., 2007). Some of these are responsible for the rapid
establishment of the body plan during the syncytial and cellular blastoderm
stages, whereas some others might be responsible for maintaining or inducing
subsequent waves of ZGA. Transcription factor-encoding transcripts are also
enriched in the pool of mRNAs present in early sea urchin embryos
(Samanta et al., 2006); these
are expressed in distinct waves at the two-cell stage, the early blastula
stage, the early gastrula stage and beyond
(Wei et al., 2006).
Interestingly, in the sea urchin, although transcripts that encode signaling
receptors are present in the maternal pool, those encoding their ligands are
strictly zygotic (Wei et al.,
2006), probably because the latter are expressed in a spatially
regulated manner to specify cell fate and position in the embryo. As mentioned
above, in the mouse, ZGA transcripts are enriched for genes involved in RNA
transactions (Hamatani et al.,
2004; Zeng et al.,
2004).

Mechanism and timing of ZGA onset

Four mechanisms that are not mutually exclusive could account for the
timing of ZGA onset.

Nucleocytoplasmic ratio

The effects of the nucleocytoplasmic ratio on the control of the embryonic
cell cycle have been studied for more than a century (reviewed by
Masui and Wang, 1998). The
nucleocytoplasmic ratio model hypothesizes that a ZGA repressor is present in
the cytoplasm of the early embryo, but is titrated by the increasing number of
nuclei (or amount of chromatin) relative to the unchanging volume of
cytoplasm. This model was proposed when it was discovered that, in polyspermic
X. laevis embryos, zygotic transcription is activated two cleavage
divisions earlier than normal (Newport and
Kirschner, 1982a; Newport and
Kirschner, 1982b). These studies also demonstrated that the timing
of X. laevis ZGA is not based on a specific number of cleavages,
rounds of DNA replication or a `clock' mechanism. Support for a
nucleocytoplasmic ratio model also comes from zebrafish, where a mutation that
blocks chromosome segregation but leaves cell division unaffected results in
polyploid cells (with a very high local DNA concentration) in which
transcription starts several cycles earlier than in wild-type cells
(Dekens et al., 2003).

Box 3. Genome-wide expression profiling: some caveats

At any given point during the MZT, both maternal and zygotic versions of
the same transcript can exist. In general, standard microarray-based analyses
(or `deep-sequencing' methods) cannot distinguish between these two types of
transcript. This is particularly problematic when assessing both the extent
and the mode of maternal transcript degradation, as well as the extent of
zygotic transcription. For example, the observation that a transcript does not
change in abundance from the start to the end of the MZT could suggest that it
is a maternally contributed, stable mRNA. Another explanation, however, is
that the maternally loaded transcript is destabilized but is replaced by
zygotic transcription. In D. melanogaster, it has been possible to
circumvent this difficulty by using large chromosomal deficiencies to remove
simultaneously up to 40% of the genes from the embryo but not from the mother,
thus eliminating the zygotic component of the mRNA signals read for these
genes (De Renzis et al.,
2007). This study convincingly showed that two thirds of zygotic
transcripts also had a maternal contribution that would otherwise have masked
their synthesis during ZGA. With respect to the degradation of maternally
supplied transcripts, genome-wide expression profiling studies usually do not
have enough temporal resolution to determine whether any specific maternal
mRNA is eliminated by maternal and/or by zygotic degradation pathways (see
text). This problem can be circumvented in part by removing the latter pathway
either through the use of transcriptional inhibitors or, in D.
melanogaster, by studying activated, unfertilized eggs in which the
degradation of maternal mRNAs occurs in the absence of zygotic transcription
(Tadros et al., 2007).

A candidate transcriptional repressor titrated by increases in chromatin
levels is the X. laevis homolog of DNA methyltransferase (xDmnt1),
the depletion of which results in premature zygotic transcription
(Stancheva and Meehan, 2000).
Surprisingly, the role of xDmnt1 as a transcriptional repressor appears to be
independent of DNA methylation, as premature transcription can be rescued by a
catalytically inactive version of the protein
(Dunican et al., 2008). In
flies, the maternally loaded transcription factor Tramtrack (TTK) is a
titratable repressor. TTK represses the transcription of the segmentation gene
fushi tarazu (ftz) during early cleavage cycles
(Brown et al., 1991;
Pritchard and Schubiger,
1996). Eliminating TTK-binding sites or reducing the amount of TTK
results in premature ftz transcription, whereas TTK overexpression
has the opposite effect. By contrast, the modulation of the nucleocytoplasmic
ratio in haploid D. melanogaster embryos does not determine the
timing of zygotic transcription (Edgar et
al., 1986). This apparent contradiction has recently been resolved
through gene expression profiling studies that show that distinct groups of
transcripts are produced during ZGA: the timing of activation of a minority
(including ftz) depends on the nucleocytoplasmic ratio, whereas most
transcripts are activated via a maternal-clock-type mechanism
(Lu et al., 2009).

Interplay between maternal transcript and protein destabilization and
zygotic genome activation. (A) In D. melanogaster,
maternally loaded transcriptional repressors (TxnR), such as TTK, are present
in early embryos to maintain transcriptional quiescence. Egg activation
initiates the PNG kinase-mediated translation of Smaug (SMG), a
sequence-specific RNA-binding protein that triggers the destabilization of a
specific subset of maternal mRNAs. The accumulation of SMG protein is a
component of the `maternal clock', the activity of which in progressively
eliminating maternal transcripts, including those that encode known
transcriptional repressors such as TTK, ultimately reduces their levels below
a threshold, thus permitting zygotic transcription to proceed. The ensuing
transcription of components involved in the zygotic degradation activity, such
as the miR-309 family of microRNAs, accelerates destabilization and
expands the repertoire of destabilized maternal transcripts. (B) In
C. elegans, an analogous scenario plays out, this time at the
post-translational rather than the post-transcriptional level, beginning with
the egg activation-triggered phosphorylation of the zinc finger factors OMA-1
and OMA-2 by the kinase MBK-2. This induces OMA-1 and OMA-2 to sequester
TAF-4, a component crucial for the assembly of the transcription factor-II D
(TFIID) and the RNA polymerase II complex, in the cytoplasm. In the absence of
TAF-4, TFIID cannot form the stable DNA-bound complex that is necessary for
transcription, which therefore does not occur for the first two cleavage
cycles. The phosphorylation of OMA-1 and OMA-2 causes their degradation by the
four-cell stage, at which point TAF-4 is free to return to the nucleus and to
mediate TFIID-mediated transcriptional activation of the zygotic genome. MBK,
minibrain kinase; OMA, oocyte maturation defective; P, phosphorylation; PNG,
Pan gu; TAF, TBP-associated transcription factor; ZGA, zygotic genome
activation.

Maternal clock

A second model postulates that a cell cycle-independent `clock' set in
motion by the events of egg activation or fertilization triggers ZGA by
activating or producing the transcriptional machinery, or by derepressing
zygotic gene transcription. In X. laevis, evidence for a maternal
clock that regulates events during the MZT came from observations that the
timing of Cyclin A and Cyclin E1 protein destruction is independent of the
nucleocytoplasmic ratio (Howe et al.,
1995; Howe and Newport,
1996). The clock requires the translation of maternal mRNAs, as
the administration of a translational inhibitor, cycloheximide, blocks the
vast majority (83%) of ZGA in mice
(Hamatani et al., 2004). One
such mRNA that must be translated during the one-cell stage to permit ZGA is
cyclin A2 (Ccna2) (Hara et al.,
2005). The role of the Cdk2-Ccna2 cell cycle complex in ZGA,
however, remains unclear.

In D. melanogaster, gene expression profiling has shown that the
majority of ZGA depends on absolute time or developmental stage rather than on
the nucleocytoplasmic ratio (Lu et al.,
2009). SMG, which is translated upon egg activation and is
required for the destabilization of the majority of maternal transcripts
(Tadros et al., 2007), is
essential for the vast majority of high-level transcription during ZGA
(Benoit et al., 2009). The
SMG-dependent destruction of maternal mRNAs could time ZGA onset
(Benoit et al., 2009)
(Fig. 4). For example, SMG is
required for the destruction of maternal ttk mRNA. Thus, during early
embryogenesis, two processes might influence the transcriptional repression by
TTK: first, as the nucleocytoplasmic ratio increases, TTK protein would be
titrated; second, the SMG-dependent destruction of ttk mRNA, along
with TTK protein turnover would lead to a progressive decrease in TTK protein
levels. Additional support for a clock mechanism comes from the live imaging
of D. melanogaster embryos in which cyclins had been depleted by RNA
interference (RNAi), thereby blocking nuclei from entering mitosis
(McCleland and O'Farrell,
2008): centrosome division continued with a period that lengthened
over successive cycles despite the constant nucleocytoplasmic ratio.

Post-translational processes might also form part of the ZGA `timer'. For
example, phosphorylation, nuclear shuttling and protein destabilization
regulate ZGA onset in C. elegans
(Guven-Ozkan et al., 2008).
TAF-4, a crucial component in the assembly of the transcription factor-II D
(TFIID) and the RNA polymerase II pre-initiation complex, is sequestered into
the cytoplasm by the zinc finger proteins OMA-1 and OMA-2 during the one- and
two-cell stages (Fig. 4). This
inhibitory activity of the OMA complex is turned on by its
fertilization-dependent phosphorylation. In a uniquely clear-cut example of
the link between maternal product clearance and the onset of ZGA, the
phosphorylation of OMA-1 and OMA-2 is carried out by MBK-2
(Guven-Ozkan et al., 2008),
the same kinase that is responsible for the degradation of many maternal
proteins (Stitzel et al.,
2006). In fact, this phosphorylation event ultimately marks OMA-1
and OMA-2 for destruction, thereby allowing ZGA to commence in the somatic
nuclei of four-cell embryos (Guven-Ozkan
et al., 2008).

Transcript abortion

A third model postulates that incomplete zygotic transcripts are aborted by
the DNA replication machinery during the rapid early cleavage cycles. This is
supported by experiments in X. laevis
(Kimelman et al., 1987) and
D. melanogaster (Edgar and
Schubiger, 1986), in which the application of inhibitors that
block the cell cycle (thus extending interphase) result in premature ZGA.
Conversely, by studying a relatively large gene, it was demonstrated that
progression through mitosis does, in fact, abort transcription
(Shermoen and O'Farrell,
1991). This model predicts that the earliest wave of ZGA in flies,
which begins when the mitotic cycles are only eight minutes long, would be
enriched for relatively short genes. Indeed, the majority of early-expressing
D. melanogaster genes lack introns and all encode small proteins
(De Renzis et al., 2007).
However, the fact that premature transcription is not elicited when the cell
cycle is blocked prior to cycle 10 (Edgar
and Schubiger, 1986) indicates that additional mechanisms function
in very early embryos to prevent premature ZGA.

Chromatin regulation

A fourth model hypothesizes that zygotic chromatin is initially not
competent for transcriptional activation (e.g. through histone modification).
The effects of chromatin modifications on the onset of ZGA have been best
studied in mice, where the first signs of transcription occur in the male
pronucleus in the middle of the one-cell stage. Immediately after
fertilization, the protamines (see Glossary,
Box 1) that densely package the
sperm DNA are replaced with maternally derived histones
(Nonchev and Tsanev, 1990)
that are more acetylated than those associated with the maternal pronucleus
(Adenot et al., 1997;
Santos et al., 2002). This
increased acetylation is responsible for the differential transcriptional
activity of the male and female pronuclei
(Ura et al., 1997). Drugs that
alter chromatin structure are able to induce premature gene expression
(Aoki et al., 1997), which
suggests that the timing of ZGA is accomplished in part by the temporal
control of chromatin modifications. BRG1 (SMARCA4 - Mouse Genome Informatics),
a component of SWI/SNF-related chromatin remodeling complexes, has been
identified as a maternal factor that regulates ZGA
(Bultman et al., 2006).
SWI/SNF complexes are recruited by transcription factors to clear nucleosomal
DNA. When BRG1 is depleted, the transcription of 30% of the genes activated
during ZGA fails.

Apart from understanding what regulates the timing of ZGA onset, even less
is known about the transcription factors that specifically mediate ZGA or the
cis-acting elements through which these factors function. In D.
melanogaster, variants of a 7-mer sequence called `TAGteam' are
overrepresented within 500 bp of the transcription start sites of the first
wave of zygotic transcripts (ten Bosch et
al., 2006). Two factors that bind TAGteam have been identified:
Bicoid Stability Factor (BSF) and Zelda (ZLD; VFL - FlyBase)
(De Renzis et al., 2007;
Liang et al., 2008). Although
the role of BSF is unclear, ZLD, a zinc-finger transcription factor, has been
shown to be required for the first wave of ZGA in D. melanogaster
embryos (Liang et al., 2008).
Interestingly, both ZLD (Liang et al.,
2008) and SMG (Benoit et al.,
2009) are required for the transcription of the miR-309
cluster, the products of which provide feedback to destabilize a subset of
maternal mRNAs (Bushati et al.,
2008). SMG and ZLD might function independently in ZGA: SMG to
eliminate transcriptional repression, and ZLD to direct transcriptional
activation.

Functions of ZGA

To assess the developmental role of ZGA, either transcription must be
inhibited with drugs or genetic manipulations must be carried out that prevent
some or all genes from being expressed after, but not before, fertilization.
The results of experiments applying transcriptional inhibitors to early
embryos have led to the conclusion that the first developmental requirements
for zygotic transcription occur significantly after the earliest detected
traces of transcription, coinciding instead with the onset of the major
wave(s) of ZGA. For example, after α-amanitin treatment, mouse embryos
cleave to the two-cell stage, but the next cleavage is blocked
(Braude et al., 1979). In
D. melanogaster, similar treatment prevents cellularization, which
normally coincides with the major ZGA wave
(Edgar and Datar, 1996).
Despite the fact that some transcription is first detected at the four-cell
stage in C. elegans embryos
(Seydoux and Fire, 1994;
Seydoux et al., 1996),
development can continue well into gastrulation - to about the sixth or
seventh cell cycle - in the presence of α-amanitin
(Edgar et al., 1994). In both
frogs (Newport and Kirschner,
1982a) and zebrafish (Zamir et
al., 1997), zygotic transcription is required for gastrulation
immediately after the MBT.

A closer examination of the effects of transcriptional inhibitors has shown
subtle earlier developmental defects. For instance, the first, minor ZGA wave
is required in D. melanogaster embryos to establish subtle
asymmetries in nuclear density along the anteroposterior axis as early as
nuclear division cycle 11 (Blankenship and
Wieschaus, 2001). Likewise, depleting the large subunit of RNA
polymerase II in C. elegans embryos using RNAi results in defects in
the division and migration of a pair of endodermal cells as early as the fifth
cell cycle (Powell-Coffman et al.,
1996).

Genetic strategies have led to the identification of specific zygotic genes
that direct the developmental processes described above. The search for these
genes has benefited from the existence of large chromosomal deficiencies and
rearrangements in organisms such as flies and worms. In D.
melanogaster, compound chromosomes (see Glossary,
Box 1) have been used to remove
the entire zygotic genome, one chromosome or chromosomal arm at a time
(Merrill et al., 1988;
Wieschaus and Sweeton, 1988).
Following the use of smaller chromosomal deficiencies, these studies have
shown that zygotic genes are not required during the early cleavage cycles and
that a surprisingly small number of zygotic loci (eight, including
nullo, which is one of the 30 earliest-expressed zygotic genes
discussed above) are essential between cycle 14 and the onset of gastrulation.
Similar experiments in worms, in which half of the zygotic genome was
systematically removed, did not recover phenotypes as severe or as early as
those observed after transcriptional inhibition
(Storfer-Glazer and Wood,
1994). This might be attributable to genetic redundancy;
alternatively, essential early-acting zygotic genes might reside in the
untested half of the genome.

Conclusions

“what's done, is done.”Shakespeare, Macbeth (III.ii)

To reach a full understanding of the mechanisms and functions of maternal
transcript destabilization and zygotic genome activation during the MZT, it
will be necessary to define the relationship of these components to each
other. For example, even though we know that zygotic transcription is required
for the degradation of a subset of maternal transcripts, it is not yet clear
whether the converse is true. We now have genome-wide catalogs of maternal and
zygotic transcripts for half-a-dozen animals, in some cases together with a
certain knowledge of transcript dynamics during the MZT. Whereas analyses of
these transcripts and their molecular and biological functions have been very
informative, it would not be surprising, given the high degree of
post-transcriptional regulation that occurs during early embryogenesis, if the
transcriptome and the proteome did not correlate well during this stage. The
regulation of maternal products at the level of protein stability occurs in
X. laevis, mice and C. elegans, where, for example, the
successful exit from meiosis requires the ubiquitin-mediated degradation of
several specific proteins upon egg activation
(Lu and Mains, 2007;
Rauh et al., 2005;
Shoji et al., 2006). Catalogs
of the proteome during the MZT are now beginning to appear
(Gouw et al., 2009); initial
results are consistent with the hypothesis that control of protein synthesis
and stability are as important for early development as control of mRNA
synthesis, stability and localization. Extensive post-translational
modifications, such as changes in phosphorylation state, also occur during egg
activation in frogs and sea urchins
(Mochida and Hunt, 2007;
Roux et al., 2006).
Ultimately, therefore, to reach a full understanding of the players involved
in the regulation of the MZT, we need to have accurate catalogs of the
transcriptome and the proteome, as well as of post-translational modifications
that are imposed on the proteome, before, during and after the MZT.

Genetic and RNAi-mediated analyses have led to significant advances in our
understanding of the function of several RNAs and proteins that regulate and
implement the MZT. However, many of these players have additional functions
either during oogenesis or at other developmental stages; their phenotypes are
thus pleiotropic and difficult to interpret. Reciprocally, genetic and
functional redundancy could make it difficult to obtain any phenotype at all
for some MZT components. RNAi-mediated knock down will be an important tool
with which to assess the role of pleiotropic or redundant players, because
this method can be used to exert temporal and spatial control on the removal
of the RNAs encoding one, or even several, proteins. However, given that many
of the maternal products that function during the MZT are loaded into the egg
not just as RNA, but also as protein, knock-down approaches are of limited use
in these cases as the elimination of the mRNA does not necessarily lead to
reduced protein levels. The development and application of methods to remove
proteins (Ditzel et al., 2003;
Levy et al., 1999) and/or
their post-translational modifications in a temporally and spatially regulated
manner will thus be essential to future studies of the MZT, which are bound to
uncover additional factors and novel mechanisms involved in this fundamental
developmental event.

Footnotes

Our research on post-transcriptional regulation in D. melanogaster
is supported by the Canadian Institutes for Health
Research.

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